The long-term objective of this research is to elucidate the gene regulatory network that controls the formation of retinal ganglion cells (RGCs). Although many of the critical regulatory events that direct a retinal progenitor cell (RPC) towards a particular cell fate have been identified, RPCs still remain "black boxes" whose intrinsic properties are only vaguely defined. The proposed experiments focus on the mechanisms by which RPCs commit to an RGC fate. Previously, two key transcription factors were shown to be critical for RGC development. The proneural bHLH factor Math5 is essential for RPC competence to become an RGC, while the POU domain factor POU4f2 (Brn3b) is genetically downstream from Math5 and is required for competent, specified RPCs to differentiate into RGCs. Gene expression profiles of math5-null and pou4f2-null retinas obtained using microarrays generated from embryonic retinal cDNAs revealed numerous extrinsic and intrinsic regulatory factors that depend on Math5.or POU4f2 for their expression. The profiling analysis led to the construction of a gene regulatory network consisting of several hierarchial layers. Genetic ablation of RGCs during embryogenesis demonstrated that they are not required for the differentiation of other retinal cell types but are necessary for regulating the number of overlying RPCs by secreting growth factors such as Sonic hedgehog. The overriding hypothesis is that RPCs are a heterogeneous population of cells whose properties are defined largely by combinations of bHLH, homeobox, and other transcription factors. The specific aims are to: (1) use genetic ablation of RGCs to identify differences in gene expression in math5-null, pou4f2-null, and RGC-ablated retinas and to create an adult mouse model for RGC loss and optic nerve degeneration;(2) manipulate the properties of math5-expressing progenitor cells by replacing Math5 with other transcriptional regulators to determine the extent to which RPCs can adopt new fates;(3) elaborate the model for the RGC gene regulatory network by identifying cis-regulatory elements and transcription factors associated with RGC-specific gene expression;and (4) determine whether the ectopic expression of sonic hedgehog in RPCs restores normal numbers of RPCs in RGC-ablated retinas. The proposed experiments will lead to a better understanding of RPC behavior, which will eventually provide the means to manipulate RPCs for the purpose of retinal regeneration.